WO2023156532A1 - Vésicules dérivées de la membrane plasmatique (pmdv) isolées destinées à être utilisées dans le traitement d'infections virales - Google Patents

Vésicules dérivées de la membrane plasmatique (pmdv) isolées destinées à être utilisées dans le traitement d'infections virales Download PDF

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Publication number
WO2023156532A1
WO2023156532A1 PCT/EP2023/053907 EP2023053907W WO2023156532A1 WO 2023156532 A1 WO2023156532 A1 WO 2023156532A1 EP 2023053907 W EP2023053907 W EP 2023053907W WO 2023156532 A1 WO2023156532 A1 WO 2023156532A1
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WIPO (PCT)
Prior art keywords
plasma membrane
derived vesicles
treatment
derived
vesicles
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PCT/EP2023/053907
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English (en)
Inventor
Javier SÁNCHEZ CÉSPEDES
Jerónimo PACHÓN DÍAZ
Elisa CORDERO MATIA
Maria Eugenia PACHÓN IBAÑEZ
Judith BERASTEGUI CABRERA
Maria Esperanza BALSERA MANZANERO
Marta Claudia CARRETERO LEDESMA
Original Assignee
Servicio Andaluz De Salud
Universidad De Sevilla
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Application filed by Servicio Andaluz De Salud, Universidad De Sevilla filed Critical Servicio Andaluz De Salud
Publication of WO2023156532A1 publication Critical patent/WO2023156532A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the present invention refers to the medical field. Particularly, the present invention refers to isolated plasma membrane-derived vesicles, such as microsomes, for use in the treatment of viral infections, wherein the plasma membrane-derived vesicles can capture viral particles from the bloodstream.
  • isolated plasma membrane-derived vesicles such as microsomes
  • Epidemics of emerging and re-emerging viral diseases such as the current pandemic by the SARS-CoV-2 pose a real threat to global health.
  • new influenza virus (IV) types SARS-CoV-2, Ebola virus (EV), Zika virus (ZKV), dengue virus (DENV), West Nile virus (WNV), Chikungunya virus (CHIKV), human adenovirus (HAdV) or herpes viruses, such as HH8 or HH6.
  • niclosamide an anthelmintic drug approved by the regulatory agencies that has been shown to have significant antiviral in vitro activity against a wide variety of viruses including IV, CHIKV, WNV, DENV, VACV, CMV, and HAdV.
  • the present invention is focused on solving this problem and a reliable strategy has been designed aimed at controlling most of the viral diseases for which nowadays there is not a therapeutic or prophylactic alternative available.
  • the present invention refers to isolated extracellular vesicles, preferably plasma membrane- derived vesicles, more preferably microsomes, for use in the treatment of viral infections, wherein the vesicles are able to capture viral particles in the bloodstream.
  • virus-vesicle complexes are removed from the patient, for instance by plasmapheresis or similar methods.
  • plasma membrane-derived vesicles like microsomes, which are membranous structures containing the same composition of the plasma membrane, can be used as “tools” for the "capture” of viral pathogens.
  • lipid membranes that make up the different cell compartments are recircularized, constituting microsomes, which are plasma membrane-derived vesicles covered by a lipid bilayer of cellular origin and which therefore have exactly the same composition as they have in their original compartment.
  • plasma membrane-derived vesicles such as microsomes
  • an external device to allow the collection of these plasma membrane-derived vesicles containing the viral particles.
  • the external device is able to retain the plasma membrane-derived vesicles (loaded with viral particles) by means of plasmapheresis, because the plasma membrane-derived vesicles have been previously magnetised (filled with magnetic nanoparticles).
  • plasma membrane-derived vesicles such as microsomes, originated from the plasma membrane of cells, contain the perfect binding motifs to attach viral pathogens (see Figure 1 and Figure 2), and their combination with an extracorporeal filtration device, for the elimination of viral pathogens from the bloodstream.
  • results provided by the present invention show, as proof-of- concept, how the infection of adenovirus (HAdV) has been blocked using a crude extract of microsomes preincubated with viral particles.
  • HdV adenovirus
  • the same strategy could be used to capture any viral particle from the bloodstream.
  • the first embodiment of the present invention refers to isolated plasma membrane-derived vesicles for use in the treatment of viral infections, characterized in that the plasma membrane-derived vesicles are able to capture viral particles from the bloodstream.
  • the present invention refers to a method for the treatment of viral infections which comprises the administration of plasma membrane-derived vesicles to the patient, wherein the plasma membrane derived vesicles are able to capture viral particles from the bloodstream.
  • the plasma membrane-derived vesicles are microsomes.
  • the plasma membrane-derived vesicles are in the form of a crude extract.
  • the plasma membrane-derived vesicles are obtained by means of a purification process, which comprises: 1) Cell extract homogenization, 2) Centrifugation and fractioning to obtain the microsomal fraction.
  • the plasma membrane-derived vesicles are derived from A549 human lung carcinoma cell line.
  • an adenovirus infection is treated.
  • an adenovirus infection caused by Human adenovirus C serotype 5 (HAdV-5) is treated.
  • an adenovirus infections caused by Human adenovirus C serotype 5 is treated and the plasma membrane-derived vesicles are derived from A549 human lung carcinoma cell line.
  • the treatment is a therapeutic treatment or prophylactic a treatment.
  • the plasma membrane-derived vesicles are magnetized because they are filled with magnetic nanoparticles.
  • the magnetised plasma membrane-derived vesicles are used in combination with an extracorporeal filtration device, for collecting the plasma membrane-derived vesicles loaded with viral pathogens from the bloodstream.
  • the second embodiment of the present invention refers to a composition (preferably a pharmaceutical composition) which comprises isolated plasma membrane-derived vesicles, such as microsomes and, optionally, pharmaceutical acceptable excipients or carriers.
  • the composition comprises a crude extract of plasma membrane-derived vesicles.
  • the composition comprises plasma membrane- derived vesicles coming from A549 human lung carcinoma cell line or from any other target cell line of the viruses to be captured.
  • the vesicles of the invention containing the viral particles can be eliminated from the bloodstream by both natural or non-natural processes.
  • the vesicles of the invention containing the viral particles will likely be cleared from blood mostly by the mononuclear phagocyte system [Biomaterials. 2021 Aug;275: 121000. doi: 10.1016/j.
  • the vesicles of the invention containing the viral particles could be eliminated for instance by apheresis (which is a procedure in which blood is collected, part of the blood comprising vesicles is taken out, and the rest of the blood is returned to the donor) or any other device such as a system suitable for treating viral infections by capturing and eliminating viral particles from the bloodstream, which comprises: a. A composition comprising magnetized plasma membrane-derived vesicles, such as microsomes, for capturing viral particles from the bloodstream; and b. Extracorporeal filtration device for collecting and eliminating the plasma membrane-derived vesicles loaded with viral pathogens from the bloodstream.
  • apheresis which is a procedure in which blood is collected, part of the blood comprising vesicles is taken out, and the rest of the blood is returned to the donor
  • any other device such as a system suitable for treating viral infections by capturing and eliminating viral particles from the bloodstream, which comprises: a. A composition comprising magnetized plasma membrane-
  • PMdV phosphatidylcholine
  • a membrane-derived vesicles are sub-membrane fragments of variable size, in the nanometre range, shed from the plasma membrane of cells during cell growth, activation, proliferation, senescence, apoptosis and when stimulated.
  • “Microsomes” are closed vesicles of between 50 and 300 nm that result from the fragmentation of the endoplasmic reticulum of liver cells obtained by ultracentrifugation of liver homogenates, which contain oxidase function that depends on cytochrome P450 and where metabolic transformations are catalysed, which are widely used for the study of drug metabolism. After the homogenization of a cell extract, the lipid membranes that make up the different cell compartments are recircularized, constituting microsomes, spherical vesicles covered by a lipid bilayer of cellular origin, and which therefore have a composition exactly the same as what they would have in their original compartment.
  • microsomes There are two types of microsomes according to their origin: those that come from the rough endoplasmic reticulum are lined with ribosomes and are called rough microsomes, and the so-called smooth microsomes, coming from parts of the smooth endoplasmic reticulum and from vesicular fragments of the plasma membrane, the Golgi apparatus, and mitochondria.
  • Figure 4 TEM images of vesicles and their association with HAdV.
  • Sucrose will be added then to the lysed cells to 30% final and spin at low speed (5 min, 200 x g, using less than 0.3 ml/tube) to remove nuclei (pellet). Supernatant will be spin again for 2 min at 200 x g, to get rid of any rest of nuclei. Then, the supernatant will be diluted with an equal volume of water (final sucrose concentration 15%), and the total volume will be divided into tubes with ⁇ 0.3 ml/tube and spin at top speed (16000-21000 x g) for 30 min. At this point, the pellet will contain our total vesicles solution that will be washed twice with 0.5 ml Tris/EDTA buffer and spin for 10 min top speed.
  • Example 1.2 Generation of PMdV pre-filled with nanoparticles of magnetite.
  • Nanoparticles of magnetite stabilized with anionic coating will be purchased from FerroTec (Bedford, NH, USA). The particles will have a nominal diameter of 10 nm, determined by TEM, a coefficient of viscosity of less than 5 mPa s at 27°C, and a 1.8% volume content in magnetite. These nanoparticles will be added to the solution of whole cells during the process for PMdV generation protocol, just before the cycles of freeze-thaw (liquid nitrogen + water bath at 37°C) to break cells. That way, when we crash the cells and let the PM fragments to form microsomes, these will end up recirculate keeping inside them the medium containing the nanoparticles of magnetite.
  • Example 1.3 Recovery of the PMdV pre-filled with magnetite and virus complexes.
  • Example 1.4 Design and development of an extracorporeal device to assess the recovery of the prefilled with nano-magnetic particles PMdV.
  • A549 human lung carcinoma cell line was used to generate a crude microsome solution.
  • the pathogen used was HAdV5-GFP, which has the coxsackie-adenovirus (CAR) receptors as specific receptors, present on the plasma membrane of A549 cells.
  • CAR coxsackie-adenovirus
  • a solution containing the isolated microsomes was pre-incubated at 37°C for 1 h with the HAdV particles.
  • the preincubated solution with the virus was subsequently added to a cell culture plate at a MOI of 2000 pv/cell.
  • an infection control the same concentration of viral particles was used in the same diluent where the generated microsomes were resuspended and subjected to the same incubation.
  • the cell culture infected with the virus pre-incubated with the microsomes solution showed a practically total reduction in infection compared to the positive control.
  • the microsome by containing specific receptors for this virus in its membrane, could act as an anchoring surface for HAdV, so that they would interfere competitively with the cells of the cell culture in their uptake.
  • the virus could stay anchored to the microsomal surface and, in this situation, the subsequent binding and internalization processes in the host cells may be altered because of the structural impairment resulting from this conformation.
  • the interaction of the virus particles with the microsomes could result in the stable capture and internalization of the viral particle inside this vesicle after anchoring to its cell receptors, thus avoiding the infection of the cell culture by the viral particles "captured” inside the microsomes.
  • microsomes The management of disseminated viral infections using microsomes is presented as an easily accessible tool, taking into account the simplicity of the protocol for its generation and its origin, cell cultures. Moreover, the use of microsomes to control viral infections has the potential to be applied to any viral pathogen since in each case microsomes will be generated from the optimal target cells of these viruses. In addition, microsomes filled with nano- magnetic particles can be generated, which will allow their recovery from the bloodstream once the viral particles have been stably bound to them, or with an antiviral drug, to improve its pharmacokinetics.
  • Example 2.2 Extracorporeal filtration device
  • a specific system or device can be used for capturing and eliminating viral PMdV (loaded with viral particles) from the bloodstream.
  • an extracorporeal microfluidic device incorporating a flow channel design, can be used to cleanse pathogens from the flowing blood of patients with viral infections when combined with magnetized PMdV.
  • the microfluidic device comprises two adjacent, rectangular fluidic channels. One acts like a vascular channel that contains flowing blood, and the other one contains saline under intermittent or slow flow.
  • the device contains a series of open rectangular slits that will provide direct access between the blood channel and the saline-filled sinusoid channel.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
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  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des vésicules dérivées de la membrane plasmatique isolées, telles que des microsomes, destinées à être utilisées dans le traitement d'infections virales, les vésicules dérivées de la membrane plasmatique étant capables de capturer les particules virales et de les éliminer de la circulation sanguine.
PCT/EP2023/053907 2022-02-18 2023-02-16 Vésicules dérivées de la membrane plasmatique (pmdv) isolées destinées à être utilisées dans le traitement d'infections virales WO2023156532A1 (fr)

Applications Claiming Priority (2)

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EP22382139.8 2022-02-18
EP22382139 2022-02-18

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1648498A1 (fr) * 2003-08-01 2006-04-26 QUEEN MARY & WESTFIELD COLLEGE Vaccin microsomal
WO2011106376A2 (fr) * 2010-02-23 2011-09-01 The General Hospital Corporation Utilisation de microvésicules dans le traitement d'affections médicales
WO2011147086A1 (fr) * 2010-05-26 2011-12-01 江苏命码生物科技有限公司 Microvésicules contenant des petits arn interférents, leurs procédés de préparation et leurs utilisations
EP2589377A2 (fr) * 2010-07-01 2013-05-08 Aeon Medix Inc. Microvésicules issues d'un protoplaste cellulaire et leur utilisation
WO2021209995A1 (fr) * 2020-04-13 2021-10-21 Exoprother Medical Ltd. Vésicules dérivées de cellules comprenant une protéine p53 de type sauvage pour une thérapie antivirale

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1648498A1 (fr) * 2003-08-01 2006-04-26 QUEEN MARY & WESTFIELD COLLEGE Vaccin microsomal
WO2011106376A2 (fr) * 2010-02-23 2011-09-01 The General Hospital Corporation Utilisation de microvésicules dans le traitement d'affections médicales
WO2011147086A1 (fr) * 2010-05-26 2011-12-01 江苏命码生物科技有限公司 Microvésicules contenant des petits arn interférents, leurs procédés de préparation et leurs utilisations
EP2589377A2 (fr) * 2010-07-01 2013-05-08 Aeon Medix Inc. Microvésicules issues d'un protoplaste cellulaire et leur utilisation
WO2021209995A1 (fr) * 2020-04-13 2021-10-21 Exoprother Medical Ltd. Vésicules dérivées de cellules comprenant une protéine p53 de type sauvage pour une thérapie antivirale

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